IGEM:UC-Merced/2009/Notebook/E. hydro Express

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Beginning Tuesday November 13, 2012
Beginning Tuesday November 13, 2012
-
  Phage
+
11/13/2012
-
   1. Prepared Stocks
+
   Generation P1 Phage Stock
 +
 
 +
  1. Prepared P1 Stock from Yale University
     a. LB
     a. LB
-
     b. Kanomycin to add to strain JW1228-1
+
     b. Kanamycin stock 25mg/ml to add to strain JW1228-1
         C1V1=C2V2
         C1V1=C2V2
-
       25000 ug (V1) = 50ug (5ml)
+
       25000 ug/ml (V1) = 50ug/ml (5ml)
-
       C1 = 25000 ug  --> kanomycin concentration
+
       C1 = 25000 ug/ml --> kanamycin concentration
-
       C2 = 50 ug,   V2 = 5 ml broth
+
       C2 = 50 ug/ml    
-
       25000 ug (V1) - 250 ug (ml)
+
      V2 = 5 ml broth
-
       V1 = 0.01 ml = 10 uL  --> kanomycin to add to strain JW1228-1  
+
       25000 ug/ml (V1) = 250 ug
 +
      V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1
 +
      Incubate kanomycin in LB broth until 11am 11/14/12
 +
 
 +
 
 +
11/14/12
 +
 
 +
P1 Phage
 +
 
 +
1. Overnight culture did not grow. Possible reason is: The JW1228-1 culture taken from the 4°C refrigerator was old and was probably no longer living. To address this possibility we will redo the procedure using frozen JW1228-1 stock.
 +
2. Prepared new overnight culture with frozen JW1228-1 glycerol stock (following the procedure carried out on 10/13/12)
 +
    a. LB
 +
    b. Kanamycin stock 25mg/ml to add to strain JW1228-1
 +
        C1V1=C2V2
 +
      25000 ug/ml (V1) = 50ug/ml (5ml)
 +
      C1 = 25000 ug/ml  --> kanamycin concentration
 +
      C2 = 50 ug/ml 
 +
      V2 = 5 ml broth
 +
      25000 ug/ml (V1) = 250 ug
 +
       V1 = 0.01 ml = 10 uL  --> kanamycin to add to strain JW1228-1  
 +
      Incubate kanomycin in LB broth until 10:30 am 11/15/12
 +
 
 +
11/15/12
 +
 
 +
1. JW1228-1 overnight culture grew. The next step in the procedure was carried out according to Yale University.
 +
 
 +
2. Preparation of 10ml containing 20% Glucose solution
 +
    a. Calculations
 +
        20g/100 ml = x/10ml
 +
        x=2 g of glucose
 +
 
 +
3. The 20% glucose solution was made using filtered water and 2 g of glucose. The resulting 10 ml of glucose solution was filtered.
 +
 
 +
4. Preparation of Overnight JW1228-1 culture to be used in P1 phage infection
 +
    a. Calculation to make a 1:100 dilution of JW1228-1 overnight culture
 +
          1/100=x/5ml
 +
          100x=5ml
 +
          x=0.05 ml or 50 ul
 +
    b. Two culture tubes were created for P1 phage infection
 +
              i. Each tube include: 50 ul of overnight culture, 50 ul of 20% glucose, and 25 ml of 1M CaCl2
 +
5. JW1228-1 overnight culture prepared for P1 phage solution to be incubated until 11:55 on 11/15/12
 +
6. 20ul of P1 phage was added to  JW1228-1/P1 solution after incubation. It will be incubated until 11/15/12 at 3 pm
 +
7. JW1228-1/P1 culture was checked at 3 pm and there was a large amount of turbidity, which is opposite to what we should have seen. This means that potentially the P1 phage did not infect the JW1228-1 cells and instead, the JW1228-1 cells continued to grow through incubation. Due to this observation the JW1228-1/P1 culture was left to incubate until 4:30 pm on 11/15/12.
-
   
+
8. JW1228-1/P1 culture was removed from the incubator at 4:36 pm on 11/15/12. The JW1228-1/P1 culture appeared less turbid then when checked at 3 pm however, still more turbid then expected due to P1 phage activity.
 +
9. Drops of chloroform were added to the JW1228-1/P1 culture and 3 ml of solution was centrifuged in each culture tube. Only 2 ml of supernatant was available from each 35 ml centrifuge tube. The supernatant was kept in 1 ml tubes and four tubes were collected.
 +
10. The 1 ml tubes are labeled as CH AB 11/15 and kept in the 4°C refrigerator.
 +
11/28/12
-
|-
+
[Parts Submission/Transformation]
-
|colspan="2" style="background-color: #F2F2F2;"|
+
1. Plated FMJ39 (5ul) with Streptomycin (20ul), for mini prep 11/29/12
-
{{LnNotebookRecentChanges2}}
+
-
|}
+
 +
11/29/12
-
__NOEDITSECTION__
+
1. FMJ39 plate showed transformation through appearance of red spotting along edge of one quadrant of plate.
-
__NOTOC__
+
2. Mini prep performed on DNA. Tube labeled "FMJ DNA" placed in -20 degrees C freezer.
 +
3. Insufficient TAE buffer to run gel - will reattempt tomorrow 11/30/12.

Revision as of 23:07, 29 November 2012

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Lab Notebook

Beginning Tuesday November 13, 2012

11/13/2012

 Generation P1 Phage Stock
  1. Prepared P1 Stock from Yale University
   a. LB
   b. Kanamycin stock 25mg/ml to add to strain JW1228-1
       C1V1=C2V2
      25000 ug/ml (V1) = 50ug/ml (5ml)
      C1 = 25000 ug/ml  --> kanamycin concentration
      C2 = 50 ug/ml   
      V2 = 5 ml broth
      25000 ug/ml (V1) = 250 ug
      V1 = 0.01 ml = 10 uL   --> kanamycin to add to strain JW1228-1 
     Incubate kanomycin in LB broth until 11am 11/14/12


11/14/12

P1 Phage

1. Overnight culture did not grow. Possible reason is: The JW1228-1 culture taken from the 4°C refrigerator was old and was probably no longer living. To address this possibility we will redo the procedure using frozen JW1228-1 stock. 2. Prepared new overnight culture with frozen JW1228-1 glycerol stock (following the procedure carried out on 10/13/12)

   a. LB
   b. Kanamycin stock 25mg/ml to add to strain JW1228-1
       C1V1=C2V2
      25000 ug/ml (V1) = 50ug/ml (5ml)
      C1 = 25000 ug/ml  --> kanamycin concentration
      C2 = 50 ug/ml   
      V2 = 5 ml broth
      25000 ug/ml (V1) = 250 ug
      V1 = 0.01 ml = 10 uL   --> kanamycin to add to strain JW1228-1 
     Incubate kanomycin in LB broth until 10:30 am 11/15/12

11/15/12

1. JW1228-1 overnight culture grew. The next step in the procedure was carried out according to Yale University.

2. Preparation of 10ml containing 20% Glucose solution

    a. Calculations
        20g/100 ml = x/10ml
        x=2 g of glucose

3. The 20% glucose solution was made using filtered water and 2 g of glucose. The resulting 10 ml of glucose solution was filtered.

4. Preparation of Overnight JW1228-1 culture to be used in P1 phage infection

    a. Calculation to make a 1:100 dilution of JW1228-1 overnight culture
         1/100=x/5ml
         100x=5ml
         x=0.05 ml or 50 ul
    b. Two culture tubes were created for P1 phage infection
              i. Each tube include: 50 ul of overnight culture, 50 ul of 20% glucose, and 25 ml of 1M CaCl2

5. JW1228-1 overnight culture prepared for P1 phage solution to be incubated until 11:55 on 11/15/12

6. 20ul of P1 phage was added to JW1228-1/P1 solution after incubation. It will be incubated until 11/15/12 at 3 pm

7. JW1228-1/P1 culture was checked at 3 pm and there was a large amount of turbidity, which is opposite to what we should have seen. This means that potentially the P1 phage did not infect the JW1228-1 cells and instead, the JW1228-1 cells continued to grow through incubation. Due to this observation the JW1228-1/P1 culture was left to incubate until 4:30 pm on 11/15/12.

8. JW1228-1/P1 culture was removed from the incubator at 4:36 pm on 11/15/12. The JW1228-1/P1 culture appeared less turbid then when checked at 3 pm however, still more turbid then expected due to P1 phage activity.

9. Drops of chloroform were added to the JW1228-1/P1 culture and 3 ml of solution was centrifuged in each culture tube. Only 2 ml of supernatant was available from each 35 ml centrifuge tube. The supernatant was kept in 1 ml tubes and four tubes were collected.

10. The 1 ml tubes are labeled as CH AB 11/15 and kept in the 4°C refrigerator.


11/28/12

[Parts Submission/Transformation] 1. Plated FMJ39 (5ul) with Streptomycin (20ul), for mini prep 11/29/12

11/29/12

1. FMJ39 plate showed transformation through appearance of red spotting along edge of one quadrant of plate. 2. Mini prep performed on DNA. Tube labeled "FMJ DNA" placed in -20 degrees C freezer. 3. Insufficient TAE buffer to run gel - will reattempt tomorrow 11/30/12.

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